Interstitial axon branching is an important but little understood mechanism for establishing connections in the developing brain. During development, growth cones, the highly motile tips that explore the environment, direct growing axons into appropriate targets. However, in some tracts of the cerebral cortex, such as the corpus callosum and corticospinal tract, collateral branches extend interstitially from the axon shaft to innervate targets. In previous studies from the principal investigator's laboratory, growth cones of cortical axons were found to undergo pausing and reorganization, leaving behind active regions on the axon from which interstitial branches later developed. Local fragmentation of microtubules was also found to occur at these branch points. The goal of the work in this proposal is to understand the cellular mechanisms that underlie development of interstitial axon branches. In the first specific aim, the influence of target derived cues on branching will be studies by applying FGF-2 either by bath application or locally in the form of FGF coated beads to dissociated cultures of developing sensorimotor cortex. Effects on axon outgrowth, growth cones, and branch numbers and locations will be studied by imaging cortical neurons for extended periods of time. In the second aim, reorganization of the cytoskeleton will be studied with high-resolution digital imaging during spontaneous and FGF induced cortical axon branching. Effects on the cytoskeleton and development of branches will also be studied after application of biochemical reagents that experimentally perturb microtubules and actin filaments. To analyze dynamic cytoskeletal changes in developing axon branches and in reorganizing growth cones, microtubules and actin filaments will be labeled by microinjecting cortical neurons with fluorescent probes. Local changes in intracellular calcium levels will also be monitored during branching under normal and experimental conditions. Taken together this work will elucidate mechanisms of axon branching critical for the formation of appropriate connections during development. Results from this work may also have important clinical implications for regenerative sprouting after injury.